Plant pathogens cause considerable damage to agricultural plants every year. In soybeans alone it is estimated that plant pathogens account for, on average, a reduced yield of over 400 million bushels per year. Plant root pathogens typically account for over 40% of that loss (nearly 170 million bushels per year). Nearly all plants are susceptible to root pathogens and suffer damage resulting in yield loss from these pathogens.
One example of a plant root pathogen which causes significant crop damage is soybean cyst nematode (SCN), Heterodera glycines. SCN is a small plant-parasitic roundworm which preferentially attacks soybeans. SCN has been known to cause significant damage to soybean fields and is responsible for, on average, over 150 million bushels worth of damage to soy crops.
The second stage juvenile of SCN is the only stage which is able to penetrate roots. After entering the root, the SCN moves to the vascular tissue where it begins to feed. SCN is able to induce cell division in the root which results in the formation of feeding sites for the growing SCN. Feeding females remain stationary and continue to feed. Eventually, the female becomes large enough to break through the root tissue such that they are exposed on the surface of the root.
Males remain mobile and fertilize the exposed females who begin to produce eggs. Initially eggs are contained in an egg sac outside the female's body, however, eventually the entire body of the female becomes filled with eggs and the female dies. The egg filled body of the female is called a “cyst.”
The cysts eventually dislodge from the plant and remain in the soil. The walls of the cyst provide protection for approximately 200-400 eggs stored inside. The eggs may remain in the soil for up to several years, waiting for appropriate hatching conditions.
The symptoms of SCN infection frequently appear as an oval of plant damage, elongated in the direction of tillage, with relatively sharply defined borders. The damaged plants typically are late to close in with foliage and show stunted growth and yellowing of the plants. The effects of SCN vary greatly based on soil type, plant variety, and environmental conditions. Frequently plants in light sandy soils exhibit more extensive injury than plants in heavier soils.
Another example of a plant root pathogen is the root knot nematode. Root knot nematodes belong to the genus Meloidogyne. Although there are many species of root knot nematodes, the four most common species are M. incognita, M. hapla, M. javanica, and M. arenaria. Approximately 2000 types of plants, including tomato, soy, and maize, may be infected by some form of root knot nematodes. Infection by root knot nematode has been estimated to cause up to 5% of global crop loss.
When soil conditions are favorable (generally greater than 50° F.) and host plants are being grown, the root knot nematode will begin to grow. After reaching the second stage juvenile phase, the nematodes will hatch from their eggs and move through the soil to find plant roots. Upon reaching a root, the nematode penetrates the root and begins feeding.
The nematode causes the cells in the plant parenchyma to become multi-nucleate. These cells become “giant cells” which the nematode uses as a feeding site. The site also turns into a gall, which is a protrusion of the root in which the nematode develops.
After several molts the female is ready to begin laying eggs. The female deposits eggs in a gelatinous matrix which serves to protect the eggs. Eventually, the egg masses dissociate from the plant and remain in the soil. The eggs then begin development once conditions are favorable for hatching.
A number of techniques are being pursued in order to limit the damage by plant pathogens in general and plant root pathogens in particular. Approaches include rotating crops, the use of pesticides, and increasing plant resistance through traditional breeding and genetic manipulation.
In order to effectively use these techniques, researchers must have the ability to determine the pathogen's ability to survive and thrive on the plant. This measure of the pathogen's ability to survive on plant tissue is referred to as the pathogenicity of the pathogen. Using an accurate and sensitive measure of pathogenicity, researchers can then effectively compare the resistance of different plants, the pathogenicity of various pathogens, and select plants for breeding or manipulation to increase plant resistance.
One example of an assay of plant damage is the hairy root assay. In this assay, transgenic roots are created which have fine fibrous roots. This type of root can be generated through the use of a number of plant pathogens including Agrobactreium rhizogenes and Agrobacterium tumefaciens. The hairy roots are then subjected to a plant pathogen of interest and pathogenicity is measured. Various measures of pathogenicity exist including counting the number of parasites which survive exposure and direct assays of root damage (e.g. root weight).
A significant problem with the current assay technique is that the measures of pathogenicity are so low that background noise can sometimes drown out a treatment effect. As a result, researchers are left with using ever higher numbers of replicates to decrease the noise or face the real possibility of going forward with ambiguous results. (Plovie, et. al., Nematology, 2003, Vol. 5(6), 831-841)
Thus there exists a need in the art for increasing the sensitivity of assays for pathogenicity.